Evaluation of carotenoid production potential by Rhodococci
Rhodococci strains are well known for their versatility to utilize various compounds as substrates, such as carbohydrates, aromatic compounds, and fatty acids. In this study, four Rhodococci strains, namely R. opacus (NRRL B-3311), R. rhodochrous (NRRL B-16536), R. erythropolis (NRRL B-16531), and R. jostii RHA1 were first tested for their capability of utilizing lignin model compounds. The four strains were challenged by growing in medium containing 2 g/L sodium benzoate as the sole carbon source. Benzoate was selected because it is commonly used as lignin model compound [10, 11]. As shown in Fig. 1, among the four tested strains, R. jostii RHA1 and R. rhochrous showed fast growth, and both reached the stationary phase within 24 h of cultivation. In contrast, a 48 h lag phase was noticed for R.opacus and R. erythropolis, and R. opacus only showed a slight growth even after 96 h fermentation. R. jostii RHA1 has been reported by many studies for their high capability to utilize lignin or lignin rich liquor , but R. rhodochrous has rarely been reported to utilize lignin model compounds. Given that more robust growth was demonstrated by R. jostii RHA1 and R. rhodochrous, these two strains were selected for further carotenoid fermentation. After 96 h of fermentation in benzoate-containing medium, R. rhodochrous produced ~ 0.5 mg/L carotenoid, while R. jostii RHA1 only produced half of that. Taking into account the performance in both utilization of lignin model compounds and production of carotenoids, R. rhodochrous was chosen for the further fermentation study as discussed below.
Utilization of lignin model compounds and lignin by R. rhodochrous
Heterogenous nature of lignin often results in diverse monomers after its depolymerization. Thus, it is important to know if R. rhodochrous is also able to utilize a wide range of lignin derived compounds. To this end, we further evaluated its ability to utilize lignin model compounds other than benzoate. Vanillic acid and 4-hyrdoxybenzoic acid were selected as they are common compounds derived from lignin depolymerization [12, 13], and were reported to be used as lignin model compounds by prior studies . R. rhodochrous was able to use all the three tested lignin model compounds, but different growth patterns were demonstrated. The strain grew faster in benzoate, and reached the stationary phase within 12 h (Fig. 3), while it reached the stationary phase after 24 h of cultivation with either vanillic acid or 4-hydroxybenzoic acid. In Rhodococci, benzoate was converted into catechol, which undergoes ring opening via either ortho-cleavage or meta-pathway . Different from benzoate, 4-hydroxybenzoic acid and vanillic acid are transformed into protocatechuate, followed by ring cleavage via a series of enzymatic reactions . The similarity of catabolic pathways for vanillic acid and 4-hydroxybenzoic acid may explain the similar growth trend of R. rhodochrous when fed with these two compounds. Lastly, we tested whether R. rhodochrous can grow in medium with lignin as the sole carbon source. With 10 g/L alkaline lignin as carbon source, R. rhodochrous also showed a 7-fold increase of cellular biomass, reaching ~108 CFU/mL after 72 h of cultivation. This corresponded to an OD of ~0.3 (OD of 0.05 corresponding to about 1.5×107 CFU/mL), which is much lower than that obtained from lignin model compounds. Prior studies have proved that the use of lignin depolymerization compounds generated instead of lignin as a polymeric substrate can favor the growth of R. opacus, which in turn increase lipid production [17, 18]. Hence, it would be necessary to depolymerize lignin via either chemical or biological methods to facilitate the growth of R. rhodochrous in lignin as carbon source and improve the production of target products.
Optimization of carotenoid production
Microbial production of carotenoid is dependent on various factors, including pH, temperature, C/N ratio, and osmotic pressure [19, 20]. Undoubtedly, understanding how these factors influences carotenoid production by R. rhodochrous is critical for maximizing carotenoid biosynthesis.
To investigate the effects of C/N ratio on carotenoid production, a C/N ratio ranging from 20 to 110 was tested. With a C/N ratio of 20, carotenoid production plateaued at 120 h and decreased thereafter, while carotenoid production showed a gradual increase with the extension of culture time at a higher C/N ratio (Fig. 5b). By increasing C/N ratio to 50 and 80, an increase of 40% was observed (Fig. 5b). It is known that a high C/N stimulates lipid accumulation in cells, and carotenoids are generally hydrophobic. Thus, increased lipid contents may provide more hydrophobic regions for carotenoids to accumulates . Indeed, with the increase of C/N ratio, both lipid titer and lipid content demonstrated a concomitant increase (Fig.5c). With a C/N ratio of 50 and 80, lipid contents were about 18% and 20%, which were 5% and 7% higher than that with a C/N ratio of 20. A recent study also indicated an engineered Y. lipolytica with high capability of producing carotenoid would store produced carotenoid in lipid droplets within cells . In addition, Yamane et al. proposed that a high C/N ratio possibly reduces the consumption of NADPH for primary metabolism such as protein synthesis, leaving more NADPH available for carotenoids biosynthesis . Therefore, increased carotenoid production at higher C/N ratio could be partially attributed to increased NADPH and lipid accumulation in cells. Further increasing the C/N ratio to 110 did not improve carotenoid production despite that it had the highest lipid content (Fig.5b&c). Although biosynthetic pathways of fatty acids and carotenoids are suggested to share the same precursor (i.e., acetyl-CoA) , a high C/N ratio can flux more precursors toward lipid biosynthesis than carotenoid biosynthesis. Overall, these results indicated that an appropriate C/N ratio is critical for lipid synthesis and carotenoid accumulation. In the case of carotenoid production by R. rhodochrous, the optimum C/N ratio was 50, which was used for the latter sections.
To investigate the effects of osmotic pressure on carotenoid production, the fermentation media were supplemented with 0, 5, 15, and 30 g/L NaCl, respectively. With low concentration of NaCl (5 g/L), R. rhodochrous showed no difference in growth compared with the control where no salt was added (Fig. 6a). However, when NaCl concentration was elevated to 15 and 30 g/L, the growth rate was slowed down and less biomass was produced (Table 1 & Fig. 6a). Osmotic pressure imposed by high concentration of salt can influence microbial metabolism and growth . Although negative effects on microbial growth was incurred by osmotic pressure, carotenoid production was enhanced by ~30% when 15 g/L NaCl was supplemented compared with no NaCl addition. Further increase of salt concentration to 30 g/L retarded cell growth and had no positive effects on carotenoid accumulation. The strain’s ability to not only tolerate high salt concentration but also use salts for stimulating carotenoid accumulation is important for using lignin-based substrates. For example, the pretreatment liquor generated from alkaline pretreatment contains both lignin and salts . Such lignin-rich liquor could be directly used by R. rhodochrous to produce carotenoids, while the salts in the liquor could benefit carotenoid production. It would make the process more practical and economical while converting salt-containing lignin waste streams.
It has been reported that shifting the pH could promote the production of carotenoids . R. rhodochrous were usually reported to grow well at the neutral pH [27, 28]. Thus, the strain was cultured at the pH of 7 during the first 72 h, and then the pH was shifted to either 6 or 8 for the rest of fermentation. It was found that shifting pH did not change the cell growth, but affected the carotenoid production. A slightly alkaline condition with the pH to 8 increased the carotenoid production. However, an opposite effect was observed when the pH was shifted to 6.